title: Production of hydroxycinnamoyl anthranilates from glucose in Escherichia coli.
abstract: BACKGROUND
  Oats contain hydroxycinnamoyl anthranilates, also named avenanthramides (Avn), which have beneficial health properties because of their antioxidant, anti-inflammatory, and antiproliferative effects. The microbial production of hydroxycinnamoyl anthranilates is an eco-friendly alternative to chemical synthesis or purification from plant sources. We recently demonstrated in yeast (Saccharomyces cerevisiae) that coexpression of 4-coumarate: CoA ligase (4CL) from Arabidopsis thaliana and hydroxycinnamoyl/benzoyl-CoA/anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT) from Dianthus caryophyllusenabled the biological production of several cinnamoyl anthranilates upon feeding with anthranilate and various cinnamates. Using engineering strategies to overproduce anthranilate and hydroxycinnamates, we describe here an entire pathway for the microbial synthesis of two Avns from glucose in Escherichia coli.
  
  
  RESULTS
  We first showed that coexpression of HCBT and Nt4CL1 from tobacco in the E. coli anthranilate-accumulating strain W3110 trpD9923 allowed the production of Avn D [N-(4'-hydroxycinnamoyl)-anthranilic acid] and Avn F [N-(3',4'-dihydroxycinnamoyl)-anthranilic acid] upon feeding with p-coumarate and caffeate, respectively. Moreover, additional expression in this strain of a tyrosine ammonia-lyase from Rhodotorula glutinis (RgTAL) led to the conversion of endogenous tyrosine into p-coumarate and resulted in the production of Avn D from glucose. Second, a 135-fold improvement in Avn D titer was achieved by boosting tyrosine production using two plasmids that express the eleven genes necessary for tyrosine synthesis from erythrose 4-phosphate and phosphoenolpyruvate. Finally, expression of either the p-coumarate 3-hydroxylase Sam5 from Saccharothrix espanensis or the hydroxylase complex HpaBC from E. coli resulted in the endogenous production of caffeate and biosynthesis of Avn F.
  
  
  CONCLUSIONS
  We established a biosynthetic pathway for the microbial production of valuable hydroxycinnamoyl anthranilates from an inexpensive carbon source. The proposed pathway will serve as a platform for further engineering toward economical and sustainable bioproduction of these pharmaceuticals and other related aromatic compounds.
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA 94608, USA.
authors.forename: Aymerick
authors.initials: A
authors.lastname: Eudes
authors.forename: Darmawi
authors.initials: D
authors.lastname: Juminaga
authors.forename: Edward E K
authors.initials: EE
authors.lastname: Baidoo
authors.forename: F William
authors.initials: FW
authors.lastname: Collins
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
authors.forename: Dominique
authors.initials: D
authors.lastname: Loqué
mesh_terms: D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D000642:Ammonia-Lyases / Q000235:genetics / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology; D053898:Biosynthetic Pathways; D002109:Caffeic Acids / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000235:genetics / Q000378:metabolism; D029749:Dianthus / Q000201:enzymology; D004926:Escherichia coli / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering; D005947:Glucose / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D012248:Rhodotorula / Q000201:enzymology; D014443:Tyrosine / Q000096:biosynthesis; D062367:ortho-Aminobenzoates / Q000378:metabolism*
chemical_list: D002109:Caffeic Acids; D005656:Fungal Proteins; D010940:Plant Proteins; D062367:ortho-Aminobenzoates; D014443:Tyrosine; D006899:Mixed Function Oxygenases; C026252:4-hydroxyphenylacetate 3-monooxygenase; D000217:Acyltransferases; C116404:anthranilate N-hydroxycinnamoyl benzoyltransferase; D000642:Ammonia-Lyases; C023208:L-tyrosine ammonia-lyase; D003066:Coenzyme A Ligases; C019943:4-coumarate-CoA ligase; D005947:Glucose; C040048:caffeic acid